Dissecting the role of one neuronal RhoGEF amongst many: the Kalirin-7 null mouse

剖析一种神经元 RhoGEF 在众多神经元中的作用：Kalirin-7 null 小鼠

• 批准号: 7688612
• 负责人: RICHARD E MAINS
• 金额: $ 33.3万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7688612
• 关键词: 3' Untranslated Regions; APEX1 gene; Accounting; Actins; Adult; Affect; Alzheimer' s Disease; Anxiety; Axon; Behavior; Binding; Biochemical; Brain; Caenorhabditis elegans; Cells; Code; Communication; Coupled; Cues; Cytoskeleton; Data; Dendritic Spines; Development; Drosophila genus; DsRed; Elements; Excitatory Synapse; Exons; Family; Frequencies; Genes; Genetic; Genetic Transcription; GluR2 subunit AMPA receptor; Growth; Guanine Nucleotide Exchange Factors; Hippocampus (Brain); Hormonal Change; Human; In Vitro; Individual; Interneurons; Knockout Mice; Learning; Maintenance; Mammals; Membrane; Membrane Protein Traffic; Memory; Mental Retardation; Methods; Morphology; Mus; Mutate; Mutation; Nervous system structure; Neurons; PH Domain; Pathway interactions; Patients; Play; Prefrontal Cortex; Protein Isoforms; Proteins; RNA Splicing; Recovery; Role; Sampling; Schizophrenia; Shapes; Signal Pathway; Single Nucleotide Polymorphism; Site; Slice; Spectrin; Stimulus; Structure; Subcellular Fractions; Synapses; Synaptic Transmission; Testing; Tissues; Transcript; Variant; Vertebral column; base; behavior test; density; filamin; hippocampal pyramidal neuron; human APEX1 protein; human EMS1 protein; human Huntingtin protein; in vivo; mouse model; mutant; neuroligin 1; neuron development; platelet protein P47; promoter; public health relevance; receptor; recombinase; response; rho; rho GTP-Binding Proteins; spinophilin; synaptic function

DESCRIPTION (provided by applicant): Rho GTPases play key roles in neuronal development and in the formation and function of dendritic spines. Mental retardation is associated with deficits in individual Rho proteins, in the guanine nucleotide exchange factors (GEFs) that activate Rho proteins and in downstream targets of activated Rho proteins. Deficits in expression of Kalirin, a large, dual Rho GEF protein, are associated with increased iNOS levels in Alzheimer's disease brain and with decreased spine density in post-mortem prefrontal cortex from schizophrenic patients. In addition, Kalirin interacts with DISC1 (a candidate schizophrenia gene) and HAP1 (a Huntingtin interactor). Single nucleotide polymorphisms predict a significant number of individuals heterozygous for Kalirin function, with only one expressed copy or a normal and a mutated copy. These observations make a compelling case for analyzing mice in which expression of Kalirin can be manipulated both during development and in the adult. Mammals also express Trio, a highly homologous, but non- redundant gene. The single Kalirin/Trio gene in Drosophila and C. elegans plays an essential role within and outside of the nervous system. Based on our studies in cultured neurons, Kalirin plays a central role in axon initiation and outgrowth and in dendritic growth. Over-expression of the major adult splice variant of Kalirin, Kalirin-7, increases the formation of dendritic spines in pyramidal neurons and in normally aspiny interneurons. Reductions in the expression of Kalirin-7 and Kalirin-7 (an N-terminally truncated variant generated from a different promoter) result in deficits in spine formation and maintenance. We generated mouse models in which expression of Kalirin-7 and Kalirin-7 can be varied. Mice lacking the single exon unique to Kalirin-7/ Kalirin-7 (Kal7KO) are born at half the expected frequency, but survive to adulthood and reproduce. Mice heterozygous for this exon (Kal7+/KO) have diminished levels of Kalirin-7 and Kalirin-7 and show deficits in synaptic transmission. At the ultrastructural level, Kal7KO mice have a reduced number of normal excitatory synapses, plus many aberrant synaptic profiles not seen in normal mice. When tested in the elevated zero maze, Kal7+/KO and Kal7KO mice show a graded decrease in anxiety-like behavior. Mice in which the Kal7 exon is surrounded by lox-p sites (Kal7CKO) allow tissue-specific, developmentally regulated elimination of Kalirin-7/ Kalirin-7. These mice will be assessed using behavioral tests, morphological assessment of pre- and post-synaptic elements, electrophysiological recordings of slices and biochemical analysis of subcellular fractions. Spine formation in hippocampal neurons prepared from Kal7KO mice can be rescued by expressing exogenous Kalirin-7, allowing detailed analysis of the role of Kalirin-7 and the isolated Sec14p, spectrin-like, DH and PH domains. The role of Kal7 in spine formation in response to proteins such as Shank3, GluR2 and Neuroligin-1 will be assessed. The ability of the six known human Kalirin-7 mutants to rescue spine formation and synaptic function will be assessed using the Kal7KO mice. PUBLIC HEALTH RELEVANCE: Excitatory synapses onto dendritic spines account for much of the communication that goes on between neurons. The number and shape of dendritic spines respond to developmental cues, environmental stimuli and hormonal changes. Changes in spine morphology play key roles in learning and memory and it is clear that many signaling pathways affect spine formation and function. Kalirin-7, an activator of small GTP binding proteins of the Rho family, is localized to dendritic spines and is one of a small number of factors known to be capable of increasing the number of dendritic spines. We plan to use the Kalirin-7 knockout mouse that we generated to elucidate the pathway(s) controlling spine formation and structure.

描述（由申请人提供）：Rho GTP酶在神经元发育以及树突棘的形成和功能中发挥关键作用。精神发育迟滞与个体Rho蛋白、激活Rho蛋白的鸟嘌呤核苷酸交换因子（GEF）和激活的Rho蛋白的下游靶点中的缺陷相关。Kalirin是一种大型的双重Rho GEF蛋白，其表达缺陷与阿尔茨海默病脑中iNOS水平的增加以及精神分裂症患者死后前额皮质中棘密度的降低相关。此外，Kalirin与DISC 1（候选精神分裂症基因）和HAP 1（亨廷顿相互作用因子）相互作用。单核苷酸多态性预测了大量的Kalirin功能杂合子个体，只有一个表达拷贝或一个正常和突变拷贝。这些观察结果为分析小鼠提供了一个令人信服的案例，在这些小鼠中，Kalirin的表达在发育期间和成年期间都可以被操纵。哺乳动物也表达Trio，这是一种高度同源但非冗余的基因。在果蝇和C.线虫在神经系统内外都起着重要的作用。基于我们在培养的神经元中的研究，Kalirin在轴突起始和生长以及树突生长中起核心作用。Kalirin的主要成人剪接变体Kalirin-7的过表达增加锥体神经元和正常有刺中间神经元中树突棘的形成。Kalirin-7和Kalirin-7（由不同启动子产生的N-末端截短变体）表达的减少导致棘形成和维持的缺陷。我们产生了小鼠模型，其中Kalirin-7和Kalirin-7的表达可以变化。缺乏Kalirin-7/ Kalirin-7（Kal 7 KO）特有的单个外显子的小鼠以预期频率的一半出生，但存活至成年并繁殖。该外显子杂合的小鼠（Kal 7 +/KO）具有降低的Kalirin-7和Kalirin-7水平，并显示突触传递缺陷。在超微结构水平上，Kal 7 KO小鼠的正常兴奋性突触数量减少，加上许多在正常小鼠中看不到的异常突触特征。当在高架零迷宫中测试时，Kal 7 +/KO和Kal 7 KO小鼠显示焦虑样行为的分级降低。Kal 7外显子被lox-p位点包围的小鼠（Kal 7 CKO）允许Kalirin-7/ Kalirin-7的组织特异性发育调节消除。将使用行为测试、突触前和突触后元件的形态学评估、切片的电生理学记录和亚细胞组分的生化分析来评估这些小鼠。从Kal 7 KO小鼠制备的海马神经元中的棘形成可以通过表达外源Kalirin-7来拯救，从而允许详细分析Kalirin-7和分离的Sec 14 p、血影蛋白样、DH和PH结构域的作用。将评估Kal 7在响应于蛋白质如Shank 3、GluR 2和Neuroligin-1的棘形成中的作用。将使用Kal 7 KO小鼠评估六种已知的人Kalirin-7突变体拯救棘形成和突触功能的能力。公共卫生相关性：树突棘上的兴奋性突触负责神经元之间的大部分通信。树突棘的数量和形状对发育线索、环境刺激和激素变化做出反应。脊柱形态的变化在学习和记忆中发挥着关键作用，很明显，许多信号通路影响脊柱的形成和功能。Kalirin-7是Rho家族的小GTP结合蛋白的激活剂，定位于树突棘，并且是已知能够增加树突棘数量的少数因子之一。我们计划使用我们产生的Kalirin-7敲除小鼠来阐明控制棘形成和结构的途径。